1. Field of the Invention
The present invention relates to organic synthesis, and more particularly to a device and method for implementing cleavage and deprotection process in solid-phase nucleic acid synthesis.
2. Background of Related Art
Nucleic acid synthesis (generally referred to as "DNA synthesis") is well known. Generally, this is the process of constructing synthetic single-stranded oligonucleotide through linking of nucleotide, the basic building blocks for DNA. DNA synthesis is described generally in U.S. Pat. No. 4,458,066 issued to Caruthers et al, entitled "Process for Preparing Polynucleotides", which is incorporated by reference herein. The process described therein constructs a single-stranded oligonucleotide using one of several approaches in synthesizing DNA, namely the so-called solid-phase phosphoramidite method which generally involves the steps of deblocking/activation, coupling, capping and oxidation in each synthesis cycle for linking a building block on a solid-phase support. Further reference to this process of DNA synthesis may be found in "oligonucleotide Synthesis--A Practical Approach" edited by M. J. Gait, IRL Press, 1984, which is incorporated by reference herein; and in particular Chapter 3 therein entitled "Solid-Phase Synthesis of Oligodeoxyribonucleotide by the Phosphite-Triester Method" written by Tom Atkinson and Michael Smith.
In an automated system, the various steps are carried out by a reagent delivery system which dispenses a number of chemical reagents in a predetermined sequence in a cycle into a synthesis reaction column containing the solid-phase support, according to instructions from the system controller or computer. After the desired number of cycles have been completed, the synthesized oligonucleotide is separated from the reaction column and collected in a vial. This step is generally referred to as "cleavage". The oligonucleotide may further be subject to a step generally referred to as "deprotection" to complete isolation of the oligonucleotide.
The isolation of oligonucleotide involves the treatment of the solid bound oligonucleotide with a cleavage and/or deprotection reagent. Typically, this reagent is concentrated ammonia solution in water but can be other homogeneous or heterogeneous solutions of appropriate bases, alcohols and water. The cleavage and deprotection process is typically performed in two steps. The cleavage of the oligonucleotide is performed at room temperature for approximately one hour before decanting the mixture into a pressure-sealable vessel for extended higher temperature treatment to effect the removal of secondary protecting groups on the synthetic oligonucleotide. This two step process reduces the quantity of support related contaminants in the final isolated product.
In the case of concentrated ammonia-water, the ammonia component is highly volatile and noxious. At room temperature, a saturated solution of ammonia in water rapidly loses concentration and potency. This can lead to less efficient cleavage and deprotection. Spillage and transfer loses also increase with the current industry process standards.
The prior art synthesis reaction column has followed primarily two designs. The first design involves a double female luer in each end of the column. Luer connections always involve excess unused volume which must be filled with chemical during the synthesis and washed away after each step in the synthesis. As a result, this design requires excess chemical in both the synthesis steps and the wash steps. During post synthesis processing, the user is required to withdraw ammonia into one syringe and connect it to the column. Next a second syringe is connected at the opposite end of the column and the ammonia is pushed from one syringe to the other to accomplish cleavage. After completing cleavage the user must again draw all the ammonia into one syringe and then eject the ammonia into a vial which is then sealed and deprotection procedure is then followed. Extensive sample manipulation is thus required in the foregoing process.
A variation to the foregoing process requires using a male-to-male luer for connecting a needle to the synthesis reaction column.
A second type of column design practiced by Pharmacia Co. takes the form of a cassette that mounts between two threaded tube ferrules. This scheme reduces waste volume due to a luer mount but it involves complicated, inconvenient steps to cleave and deprotect the oligonucleotide from the cassette. For example, among other steps, the procedure involves centrifuging the cassette in a tube.